Although caries and malocclusion occur with a high prevalence in Chinese school-age children, there were no appropriate instrument to assess the oral health-related quality of life (OHRQoL) for this population. The aim of our study was to develop a Chinese (Mandarin) version of the Child Oral Health Impact Profile-Short Form 19 (COHIP-SF 19) and provide a preliminary test of its psychometric properties.The Chinese version of COHIP-SF 19 was developed through a standard translation and back translation procedure. The psychometric properties of the instrument were tested among 644 school-age children in Beijing, China, including the internal consistency, test-retest reliability, discriminant and convergent validity. A Mann-Whitney U test was used to determine the capability of the instrument to differentiate children with different caries and malocclusion outcomes. And partial Spearman correlations were used to determine the relationships between the OHRQoL scores and clinical-severity indicators and self-perceived health ratings, respectively.P ≤0.001). Girls had somewhat higher scores in the oral health and functional well-being subscales (P <0.05), while children from rural districts had lower scores than children from urban areas (P <0.05). We observed a low to moderate correlation between the overall COHIP-SF 19, subscale scores and clinical severity indicators as well as self-perceived health ratings, after adjustment for children’s age, gender, and school district .Chinese school-age children had relatively high OHRQoL scores, in spite of the fact that oral impacts were quite common (56.3%). The internal consistency and retest reliability were good to excellent with a Chronbach’s alpha of 0.81 and an intra-class correlation coefficient (ICC) of 0.77. Children who had active tooth decay or severe malocclusion had significantly lower COHIP-SF 19 scores (We confirmed satisfactory psychometric properties for the Chinese version of COHIP-SF 19 in a community sample of Chinese school-age children. The OHRQoL instrument should play a more important role in future clinical studies, epidemiological surveys and potential public health policy in China. The oral health-related quality of life (OHRQoL) is a multidimensional construct which contains a subjective assessment of an individual’s oral condition, functional and emotional well-being, expectations and satisfaction with care, and sense of self . Over thVarious instruments for the measurement of OHRQoL have been developed since 1990s . HoweverAlthough great effort has been devoted to measurement of children’s OHRQoL during the past decade all over the world ,9,14, onThe COHIP was developed to assess children’s oral-facial well-being with a wide range of age (8-15 years) and across ethnicities, health systems, and various conditions ,21. It cBecause of linguistic difference and cross-cultural issues, OHRQoL instruments must not only be translated but also validated in the target population prior to cross-cultural and cross-national adaptation -30.Our study aimed to develop an appropriate Chinese (Mandarin) version of COHIP-SF 19 and to assess the reliability and convergent and discriminant validity of the Chinese version of COHIP-SF 19 in school-age children. Furthermore, a description of the OHRQoL for the target population is presented here as well.The original English version of COHIP-SF 19 was obtained by the developer Dr. Broder  and was The Chinese version of the COHIP-SF 19 questionnaire consisted of 19 questions forming 3 conceptual subscales: oral health (5 items), functional well-being (4 items), and socio-emotional well-being (10 items). Two of the items were positively worded questions. Children were asked how often they had experienced oral impacts during the past 3 months and each question was answered with a five-point Likert scale ranging from ‘never’ to ‘almost all the time’. Responses to the two positively worded questions were recorded as ‘never’ =0, ‘almost never’ =1, ‘sometimes’ =2, ‘fairly often’ =3, and ‘almost all the time’ =4. Scoring for the 17 negatively worded items was reversed. As a result, higher COHIP-SF 19 scores reflected a more positive OHRQoL . The oveCharter [This cross-sectional study was conducted in Beijing, the People’s Republic of China, with a target population of children from 7- to 13-year-old enrolled in public primary schools. The study was approved by the ethics committee of the Peking University School and Hospital of Stomatology (No. PKUSSIRB-201412014). Nonrandom samples were used to collect data. As was suggested by Charter , the minCharter , a samplAfter describing the purpose of the study, the consent of each child and guardian was obtained prior to the study. The consent of the children had precedence over parental consent. A self-administrated questionnaire was completed by each child. Two trained research assistants were available to provide assistance for younger children, if needed. Pictures and a cue sheet were also available for younger children to help them comprehend the questions. All participants completed the questionnaire within 6-7 minutes. The same questionnaire was used 2-3 weeks later on 159 (aprroximately 25%) of the participants for the purpose of estimating retest reliability.Following completion of COHIP-SF 19, each child received a dental examination performed by one of the four calibrated dentists. The four dentists achieved satisfactory agreement prior to the project. Kappa values for the examination of caries were from 0.60 to 0.75 inter-examiner and from 0.66 to 0.88 intra-examiner. According to the intra-class correlation coefficient (ICC), inter- and intra-examiner reliability was between 0.84 to 0.94 and 0.77 to 0.92, respectively, for malocclusion examination. As the target population had different types of dentition, the number of carious deciduous (dt) and permanent (DT) teeth was recorded according to World Health Organization (WHO) criteria . The numPrior to data analysis, participants that did not complete more than 75% of responses were excluded. On the subscale level, if more than two-thirds of the items were missing in a certain subscale, the sample was catagorized as missing. If fewer items were missing, the missing values were replaced using the mean of available items.The COHIP-SF 19 impact was defined as rating 17 negatively worded items as ‘fairly often’ or ‘almost all the time’ or rating 2 positively worded items as ‘almost never’ or ‘never’.The distribution of overall COHIP-SF 19 and each subscale scores was tested using the Kolmogorov-Smirnov test.Internal consistency for COHIP-SF 19 was measured using Chronbach’s alpha, corrected item-total correlation and Chronbach’s alpha if an item was deleted. Test-retest reliability was indexed by the ICC.Discriminant validity of the scale was assessed by comparing the mean total scores across the ‘with/without active decay’ and ‘with/without orthodontic treatment need’ groups. Discriminant validity was further evaluated by examing the association between the COHIP-SF 19 scores and clinical severity with the number of decayed teeth (DT + dt) and the categories of malocclusion  adjusted by age, gender, and school district.Convergent validity was assessed by examining the relationship between COHIP-SF 19 scores and the rating of self-perceived health/oral health and dental-treatment need after controlling for demographic covariates.p <0.05.The non-parametric Mann-Whitney U test and partial Spearman correlation were used. SPSS version 20 was used for analysis. The significance level was set at In all, 644 children were selected to participate the study. Ten parents and one child declined to provide consent for a response rate 98.3%. Eight participants undergoing orthodontic treatment were excluded from the analysis procedure. Also, 5 students who were outside of the age range (less than 7 years old) were not included. Finally, of 620 participants, 53.2% (n =330) were boys between the ages of 7 to 13 with a mean age of 9.1 ± 1.5 years. Three hundred and ninety-one students (63.1%) were from rural districts. None of the cases were dropped due to incomplete data.The mean COHIP-SF 19 score was 62.2 ± 8.2 and the median was 64 (range 25-76). Mean, median, range, and quartiles for all COHIP-SF 19 responses and each subscale score are shown in Table Of all participants, 72.4% (n =449) and 44.0% (n =273) reported that their general health or oral health was excellent or good, respectively, while 43.5% (n =270) perceived that they needed dental treatment and 31.3% (n =194) reported no need for dental treatment.P <0.001) as were the subscales.The Kolmogorov-Smirnov test showed that the distribution of overall COHIP-SF 19 scores was positively skewed and significantly different from a normal distribution , and moderate for the other two subscales  and all three subscales (P ≤0.002) by the Mann-Whitney U test. Children without a need for orthodontic treatment showed significantly higher overall scores for COHIP-SF 19 and the two subscales (P ≤0.004), with the exception of the functional well-being subscale (P =0.166).The comparison of COHIP-SF 19 and each subscale score with different clinical outcomes (active decay or a normative need for orthodontic treatment) is presented in Table P ≤0.002), although the relationships were weak (│rs│ =0.12 - 0.21). Weak but statistically significant relationships were also found between the categories of malocclusion and the overall COHIP-SF 19 and two subscale scores , with the exception of the functional well-being subscale (P =0.339).Discriminant validity was further addressed by examing the relationships between clinical severity indicators and overall COHIP-SF 19 and subscale scores, after controlling for participant age, gender, and school district Table . The numP =0.026 and 0.004, respectively). Children from rural districts had significantly lower scores in the overall COHIP-SF 19 (P =0.005), the oral health subscale (P <0.001), and the functional well-being subscale (P =0.039) than participants from urban districts, but this was not true for the socio-emotional well-being subscale.Table rs =0.10). The coefficients of the partial Spearman correlations are shown in Table All of the partial correlations were significant, and all of the coefficients were positive (range =0.10 - 0.51). The highest partial correlation coefficients were between the overall COHIP-SF 19 and the oral health subscale scores and perceived oral health ratings, with values of 0.48 and 0.51, respectively. The lowest partial correlation coefficient was between the socio-emotional well-being subscale score and perceived dental treatment need  . For subBroder et al. [Asgari et al. [In the discriminant validity test, COHIP-SF 19 differentiated school-age children with different clinical indicators. It also showed that children with better dental health status had higher OHRQoL scores, as expected. In agreement with previous findings, children without active decay reported a higher OHRQoL than children with active decay in this study -26. Brodr et al. ,25. In fi et al. ,26. Altei et al. . The varConvergent validity was proven by weak to moderate positive relationships between the COHIP-SF 19 and subscales scores and subjective ratings of health/oral health and dental treatment need, in agreement with previous studies. When OHRQoL was higher, self-reported health/oral health was also higher, and the need for dental treatment was lower ,24,25. UOne of the challenges for chilren’s OHRQoL measurement is the stability of the instrument through different age groups. With the development of perception and an increase in life experience, there is potential discrepancy in OHRQoL between younger and older children . TherefoIn the discriminant validity test on other demographic variables, Chinese girls had higher mean scores in the oral health and functional well-being subscales. Korean girls also showed small but significant increases in COHIP scores . But theA limitation in the present cross-sectional study was the unbalanced and non-random sample. Although the sample size was large enough , replicaThe Chinese version of COHIP-SF 19 was successfully developed following a standard procedure for cross-cultural adaptation of an OHRQoL instrument. Meanwhile, the internal reliability, retest reliability, discriminant validity and convergent validity of the scale has been confirmed in a community sample of Chinese school-age children. With the high prevalence of oral impacts in school-age children and a lower OHRQoL among children from rural areas, the OHRQoL instrument should play a more important role in future clinical studies, epidemiological surveys and potential public health policy in China.


Natural killer T (NKT) cells are a subset of T cells that recognize glycolipid antigens presented by the CD1d protein. The initial discovery of immunostimulatory glycolipids from a marine sponge and the T cells that respond to the compounds has led to extensive research by chemists and immunologists to understand how glycolipids are recognized, possible responses by NKT cells, and the structural features of glycolipids necessary for stimulatory activity. The presence of this cell type in humans and most mammals suggests that it plays critical roles in antigen recognition and the interface between innate and adaptive immunity. Both endogenous and exogenous natural antigens for NKT cells have been identified, and it is likely that glycolipid antigens remain to be discovered. Multiple series of structurally varied glycolipids have been synthesized and tested for stimulatory activity. The structural features of glycolipids necessary for NKT cell stimulation are moderately well understood, and designed compounds have proven to be much more potent antigens than their natural counterparts. Nevertheless, control over NKT cell responses by designed glycolipids has not been optimized, and further research will be required to fully reveal the therapeutic potential of this cell type. The most studied subpopulation of NKT cells, invariant NKT cells (iNKT or Type 1 NKT cells), have a T cell receptor (TCR) with an variable (V) n humans . In addiR chains ,6.α1−α3) and a β2 microglobulin chain [CD1d, mainly expressed on B-cells, dendritic cells, macrophages, and epithelial cells, presents lipid-containing molecules to the TCR of iNKT cells . The strin chain ,7. Cd1d in chain ,9. It isin chain . For exain chain , whereasin chain ,13,14.Unlike its major histocompatibility complexes (MHC) counterparts, CD1d presents lipids, not peptides, to the TCR of iNKT cells. The groove on CD1d is narrower, deeper, and more hydrophobic than MHC class-I and II binding grooves. The antigen-binding groove forms two hydrophobic pockets, termed the A' and F' pockets. These pockets generally accommodate and bind long carbon lipid tails from a variety of lipid-containing molecules ,12. PresAfter stimulation from a CD1d-bound antigen, iNKT cells produce, within hours, large amounts of cytokines . ReleaseiNKT cells modulate innate and adaptive immune responses. Released cytokines can activate adaptive cells, such as T and B cells, and innate cells, such as dendritic cells and NK cells ,21. Thes- or β-linked to a phytosphingosine-containing ceramide backbone. The different agelasphins vary mostly by the composition and length of their lipid tails, as well as the saccharide composition. Their initial screenings demonstrated that α-linked galactose- containing agelasphin glycolipids were significantly more potent against B16 mouse melanoma cells than the β-linked agelasphins [In 1993, Kirin Pharmaceuticals, a pharmaceutical subsidiary of Kirin Brewery Inc., reported results from a systematic screening of marine natural products for anti-tumor activity. Their efforts led to the isolation of a family of glycolipids termed “agelasphins” . This SAR study also established precedence for the importance of the C3' hydroxyl on anti-tumor activity, the causality of longer ceramide acyl chains towards better anti-tumor activity, and the optimal phytosphingosine chain length (18 carbon phytosphingosine scaffold) [The promising anti-tumor activities of agelasphins, led Morita et al.  to perfocaffold) .-GalCer was realized in 1997, when α-GalCer was shown to be a CD1d-restricted iNKT cell antigen [-GalCer, became more apparent. Many well-documented studies have focused on elucidating the diseases that are affected by iNKT cells [-GalCer to see its effect on the disease in question [The importance of α antigen . ConsequKT cells . These squestion . In this-GalCer is the standard model iNKT cell antigen, it has at least two limitations that inhibit its therapeutic effectiveness. First, after iNKT cell stimulation by CD1d-bound α-GalCer, the immune system releases both TH1 and TH2 cytokines that, in some cases, can counteract one another in modulating the immune system. This was suggested by Kronenberg and coworkers [-GalCer can stimulate iNKT cells too potently causing a cytokine storm; that is, iNKT cells release a massive amount of cytokines leading to iNKT cell anergy or inactivation of iNKT cells [et al. [et al. [-GalCer challenges after administration of α-GalCer. Because of the therapeutic potential of iNKT cells and the limitations of α-GalCer, an effort to find more effective iNKT cell antigens has ensued.Although αoworkers  with theKT cells . This iN [et al.  and Pare [et al. . In bothi.e., agelasphins) are not natural antigens for iNKT cells. Consequently, substantial effort has been expended in identifying exogenous and endogenous antigens for NKT cells, and series of discoveries have revealed the breadth of iNKT cell antigens. Exogenous antigens have been identified in multiple human pathogens including bacteria, fungi, and parasites. The presence of endogenous iNKT cell antigens is implicated in the maturation of these cells and their autoreactivity, and multiple candidate antigens have been reported. In addition to natural antigens for iNKT cells, synthetic variants have been designed and prepared in efforts to modify the immunostimulatory and immunomodulatory properties of iNKT. In this review, the structures of exogenous and endogenous natural antigens will be presented. In addition, designed antigens for iNKT cells will be described.It has been widely accepted that glycolipids from marine sponges , which differ primarily from GSL-1 by the presence of additional saccharides, including the presence of a glucosamine subunit in both. GSLs share remarkable similarities to α-GalCer, especially in the α-glycosyl linkage.The KT cells ,33,34. F-GalCer. The difference in stimulatory activity of these two glycolipids likely results from protein interactions, which differ due to the stereochemistry at the C4"-position in the glycolipids . Crystal structures of the TCR-GSL1-CD1d and TCR-α-GalCer-CD1d complexes show different hydrogen-bonding networks, which result in a lateral shift of the galactosyl head group in α-GalCer relative to the glucuronosyl head group in GSL1. This lateral shift is thought to explain the difference in antigenicity between GSL-1 and α-GalCer [GSL-1 is not as strong of an iNKT cell antigen as αα-GalCer ,35,36.et al. [et al. [Sphingomonadaceae to validate proposed structures and determine their stimulatory activity with iNKT cells. These studies confirmed the previously published evidence that GSL-1 is an iNKT cell antigen. However, it was observed that synthetic forms of the higher order GSLs, GSL-3 and GSL-4, were not strong antigens for iNKT cells. This observation was unexpected for two reasons: (1) additional sugars attached to α-GalCer are enzymatically removed in the lysosome revealing antigenic α-GalCer, and it was expected that a similar process would occur with the higher ordered GSLs revealing GSL-1, and (2) in an earlier report, [-GalCer, Long et al. [As is customary in the practice of structural confirmation of isolated natural products, Long et al.  and Kinj [et al.  synthesi report,  isolatedg et al.  showed tet al. [Mycobacterium bovis bacillus, as an exogenous NKT cell antigen [et al. [These conflicting results between synthetic and isolated glycolipids are not unique to GSL-4. For example, Fischer et al.  presente antigen . However [et al.  synthesiSphingomonadaceae family, while ubiquitous, are not common human pathogens. In 2006, Kinjo et al. [Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease affects an estimated 33,000 people each year in the U.S. making it the most common vector-borne disease in the U.S. [B. burgdorferi than wild-type mice, Kinjo et al. [ in vivo during an infection with this organism.The discovery that microbial glycolipids stimulate iNKT cells provided insight into the role of iNKT cells in innate immunity. However, it is generally accepted that bacteria from the o et al.  reportedthe U.S. . After oo et al.  demonstrB. burgdorferi, a series of galactosyl diacylglycerols (DAGs) were isolated. These glycolipids contain varied lengths of acyl chains and different degrees of unsaturation. To further ensure that the DAGs were the active iNKT cell stimulatory components in the tested extracts, Kinjo et al. [et al. [B. burgdorferi antigens to CD1d. Analysis of the crystal structure of many isoforms of DAGs bound to CD1d showed that the length and degree of saturation of the acyl chains, specifically which acyl chain is bound in the A' or F' pocket, impacts how the glycolipid is bound in CD1d. Furthermore, alternate binding motifs result in different orientations of the carbohydrate head group. The chain lengths and unsaturation found in BbGL-II provide a galactose orientation comparable to that found in the CD1d complex with α-GalCer.In the process of characterizing antigenic glycolipids in o et al.  synthesi [et al.  further et al. [Streptococcus pneumoniae. These glycolipids are similar to the DAGs found in B. burgdorferi   . Their dAspergillus spp. These fungi are logical targets since they have been reported to present significant respiratory exposure in humans [A. fumigatus and A. niger extracts, isolated and purified asperamide B, and synthetic asperamide B rapidly induced airway hyperreactivity and airway inflammation, cardinal features of allergic asthma in vitro and in vivo [A. niger [Over the past decade, substantial evidence has accumulated suggesting that iNKT cells are involved in triggering asthma. In addition, common molds have been associated with increased incidence of asthma. These observations prompted Umetsu and coworkers  to explon humans ,47. Thei in vivo . AsperamA. niger .-linked glycolipid. It also contains modified acyl and sphingosine chains, as compared to α-GalCer. The acyl chain is βδ unsaturated with an α hydroxyl group. The sphingosine chain is unsaturated with two additional methyl groups. These differences in structure may account for the recognition of this glycolipid as a β anomer rather than the more common α form seen with other iNKT cell antigens [Unlike the previously described iNKT cell exogenous antigens, asperamide B  is a β-lantigens . Helicobacter pylori, a common Gram-negative bacterium found in the stomach, infections are inversely correlated with asthma [et al. [H. pylori to suckling mice prevented allergen induced airway hyperreactivity later in their lifecycle. This compound was also shown to activate both mouse and human CD1d-restricted iNKT cells in a TH1-biased manner. Supporting the recent report that h asthma , Chang e [et al.  found thDuring iNKT cell development in the thymus, the TCR of an iNKT cell population samples low affinity endogenous antigens as part of the positive selection process. If the TCR samples a high affinity ligand or if it does not sample an endogenous ligand, then the T cell population is clonally deleted . Efforts-hexosaminadase knockout mice had a significant decrease in the number of iNKT cells. This enzyme is responsible for the truncation of GalNAc residues from a variety of lysosomal glycolipids yielding iGb3, isomeric Gb3, and other related glycosphingolipids. Synthesis of affected glycolipids and observation of iNKT cell stimulation by them led to the observation that among this group of glycolipids only iGb3 proved stimulatory [The first endogenous iNKT cell antigen reported was isoglobotrihexosylceramide (iGb3) . iGb3 wamulatory . The relmulatory , the inamulatory , and themulatory ,58. Nevemulatory ,57.et al. [In an effort to identify additional endogenous ligands, Cox et al.  performeet al. . This waet al. . Bound pet al. . Though -GalCer. GNPAT knockout mice, mice lacking a key enzyme necessary for the synthesis of these compounds, exhibit multiple abnormalities as well as decreased numbers in iNKT cells further supporting the importance of these lipids. GNPAT knockout mice had reduced numbers of iNKT cells further suggesting the presence of multiple selecting endogenous antigens for the maturation of iNKT cells.In the continuing effort to identify endogenous antigens, Libero and coworkers  extracte-glucosylceramide , as well as enhanced prevention of melanoma metastasis in mice. Because both of these disease states are related to the release of IFN-γ, these results suggested that α-C-GalCer was a better inducer of IFN-γ than α-GalCer. As expected, the cytokine release profile after α-C-GalCer administration resulted in a highly skewed TH1 bias of cytokines [-GalCer in multiple disease state models  [In an attempt to develop more potent iNKT cell antigens, groups have also modified the glycosidic linkage of αC-GalCer . Adminisytokines . Over thelanoma)  and as aelanoma) . -C-GalCer and to possibly enhance its activity, several groups have designed analogs of α-C-GalCer. Analysis of the α-GalCer-CD1d and TCR-α-GalCer- CD1d crystal structures provided two relevant features. First, the glycosidic oxygen participates in hydrogen-bonding which would be absent in the α-C-GalCer-CD1d complex. This would result in weaker binding to the CD1d-TCR complex. Second, α-GalCer is presented at a 170 degree dihedral angle, and this might be an optimal angle for presentation to the TCR interface [-C-GalCer analogs with a double bond linking the anomeric carbon of galactose to ceramide, effectively locking the sugar in place . With toduction , most maoduction , 3"- andoduction , and a poduction ,110. Unl-GalCer-CD1d crystal structure [-GalCer analog probed the ability to minimize the amount of contacts necessary for stimulation. ThrCer, a ceramide with an α-threitol chain, was able to stimulate iNKT cells without the associated iNKT cell anergy. This analog makes the necessary hydrogen-bonds with the TCR of iNKT cells, further providing evidence for the importance of the hydrogen bonds in iNKT cell stimulation [These observations were later explained from the crystal structure of the iNKT cell TCR-αtructure . The 2"-mulation . -GalCer bound to CD1d, few things were known about the structural requirements necessary for iNKT cell activation. Kronenberg and coworkers [-GalCer analogs have been an attractive target in the design of variants with diverse applications and properties.Before the availability of the crystal structure of αoworkers  showed t et al. [-GalCer. Xia et al. [ et al. [-GalCer analogs can be used as probes for the analysis of glycolipid uptake. One possible issue with these analogs is that modifying the glycolipid with relatively large fluorophores or biotin might interfere with or completely change the trafficking, processing, and presentation. To address these possible issues, Liu et al. [-GalCer analog, in a lysosomal processing model. Their studies concluded that the acetamide position did not affect lysosomal processing. The effect of the 6"-modification towards trafficking and presentation has not been fully elucidated.One such application was demonstrated by Zhou et al. , who cona et al.  and Chen[ et al.  reportedu et al.  synthesi-GalCer. For example, α-GalCer’s therapeutic effectiveness is limited by the observed iNKT cell anergy and counteracting released cytokines, but it has practical limitations because of its poor solubility in organic and aqueous solvents. To address this issue, Liu et al. [-GalCer (PBS-57  in vivo and in vitro when compared to α-GalCer.6"-Analogs have also been designed to overcome the therapeutic and practical limitations of αu et al.  incorporr PBS-57 . The incet al. [-GalCer (-GalCer accompanied with a diminished production of IL-4. This marked the first sugar-modified analog that could bias the iNKT cell response. Aspeslagh et al. [-GalCer-TCR crystal structure. This revealed that the naphthyl functional group of NU-α-GalCer sat in a small induced-fit binding pocket above the A' pocket instead of protruding into the solvent. In addition to a hydrogen bond interaction from the carbonyl oxygen from the urea substituent, the induced-fit binding pocket imparts a higher affinity of NU-α-GalCer to CD1d resulting in a longer interaction with iNKT cells and a TH1 biased response. Aspeslagh et al. [-GalCer provided superior prevention of tumor metastasis in B16 mice when compared to α-GalCer.The increased solubility and stimulatory activity of PBS-57 prompted Trappeniers et al.  to desig[-GalCer , a urea h et al.  further h et al.  also pre et al. [-GalCer analogs ( et al. [-GalCer analogs in an attempt to mirror the reported higher affinity of multimeric ligands in other biological systems. The analogs stimulated iNKT cells but did not significantly improve upon the immunological activity of α-GalCer.Similar to the 6"-amide and urea analogs, Jervis et al.  designed analogs . Using c( et al.  utilized-GalCer. In order to explore the importance of the oxygen atom in the pyranose ring, Tashiro et al. [-GalCer (RCAI-56, -GalCer. The follow up immunological evaluation of RCAI-56 confirmed that RCAI-56 is a strong TH1 iNKT cell agonist that increases IFN-γ production through increased IL-12 production [-C-GalCer, RCAI-56 benefits from an ether linkage instead of the acetal glycosidic bond resulting in less susceptibility towards enzymatic degradation. Unlike α-C-GalCer, RCAI-56 maintains the hydrogen bonding capabilities of the glycosidic bond towards binding to CD1d [Another saccharide modification that has received significant attention has been the design of carbo- cyclic analogs of αo et al.  synthesiRCAI-56, . Prelimioduction . Similar to CD1d . The str to CD1d .et al. [-GalCer, aminocyclitol HS44 (in vitro and displayed a similar cytokine release profile than OCH, albeit less potently. Unexpectedly, HS44 induced similar levels of IFN-γ to α-GalCer with nearly baseline levels of other cytokine released in vivo. This in vivo TH1 response was supported by the ability of HS44 to efficiently induce an antitumor response in B16 mice. Structural analysis of the mCD1d-HS44-iNKT TCR complex revealed diminished interactions with the TCR, accounting for the decrease in potency and binding affinity of HS44. This report is another example that binding affinity does not have a direct correlation to proliferation [Similar to RCAI-56, Harrak et al.  synthesitol HS44 . This anferation . Identification of natural antigens has focused on endogenous and exogenous antigens, and it is likely that many of these have yet to be discovered. iNKT cell selection and iNKT cell autoreactivity argue for endogenous antigens, and these may play a key roles in regulating both innate and adaptive immune responses. A complete understanding of the structures and functions of endogenous antigens will likely allow manipulation of their production as a means of indirectly controlling iNKT cell activation. Exogenous antigens have been found in bacterial, fungal, and parasitic pathogens. A relatively small subset of organisms has been surveyed for iNKT cell antigens, and the search for exogenous antigens promises to continue to be a fruitful avenue of research.The structures of designed glycolipid antigens for NKT cells can be conceptually divided into four portions: lipid chains, sphingosine/ceramide head group, the glycosidic bond, and the nature of the sugar. Structure-activity studies with glycolipids and iNKT cells have focused primarily on controlling the cytokine release profiles of iNKT cells in response to glycolipid stimulation. Multiple classes of glycolipid antigens effectively trigger TH1 responses from iNKT cells, and these responses have proven effective in adjuvanting vaccines and treating some forms of cancer. It is likely that these inflammatory responses would also beneficially impact responses to infection. Fewer antigens have been discovered that trigger TH2, or immunomodulatory responses. Activation of iNKT cells by this type of antigen may be generally useful in decreasing inflammation and ameliorating autoimmune diseases.For years, the paradigm of antigen recognition by T cells involved MHC and peptide antigens. The discovery that a subset of T cells recognized glycolipids opened a new area for discovery and possible manipulation of immune responses. Observations of the roles that iNKT cells play in controlling bacterial, fungal, parasitic and viral infections and in influencing autoimmune diseases has heightened interest in natural and synthetic antigens for these cells. Research in this area has involved productive collaborations between synthetic organic chemists and immunologist and has resulted in discoveries of a series of natural and synthetic antigens for NKT cells. Continued collaborations between chemists and immunologists in this field will likely provide glycolipid antigens for iNKT cells, and potential applications range from use in improving the effectiveness of vaccines to treatment of infection to elimination of tumors to suppressing autoimmune diseases.


Origanum vulgare on HCT-116 and MDA-MB-231 cell line in vitro. In order to determine the cytotoxic effects we used an MTT viability assay. The results showed that cell growth is significantly lower in extract treated cells compared to untreated control. The effect of inhibition of cell growth was higher in the treatment of HCT-116 cell line than in MDA-MB-231. Based on the results it is determined that O. vulgare is a significant source of biologically active substances that have cytotoxic and antiproliferative activity in vitro.In the present investigation, we examined the cytotoxic effect of methanolic extract from  Origanum vulgare L. is a perennial herbaceous plant belonging to the family Lamiaceae  and breast cancer (MDA-MB-231) have been selected for the research as they are leading causes of cancer death.The aim of this study was to examine cytotoxic activity of methanolic extracts obtained from Cancer-related research is conducted worldwide every day, since cancer is a leading cause of death. These studies often involve the investigation of the effects of biologically active substances on cancer cells, and they frequently originate from plants . There iO. vulgare (in a concentration of 1–500 µg/mL) in two cell lines (HCT-116 and MDA-MB-231), using MTT assay. A dose-dependent MTT reduction (or color change from yellow to purple) was observed in both extract-treated cell lines. This experiment investigated the cytotoxic activity of the methanolic extract of p < 0.05) when compared to untreated control cells. After 24 h of treatment, higher concentrations (250 and 500 µg/mL) killed more than 50% of cells. After 72 h of treatment cytotoxic effect of those concentrations was higher, but lower concentrations had weaker cytotoxic effects than after 24 h. Maximum inhibition of proliferation was achieved after 72 h at the highest concentration (500 µg/mL).The shape of the curve shows significant inhibition of cell proliferation in HCT-116 cell line  in a dosSignificantly lower cytotoxic activity was also reported in the treatment of MDA-MB-231 cell lines , when coO. vulgare on HCT-116 cell lines than on MDA-MB-231 cells. Only 10% of the HCT-116 cells remained viable 72 h after the treatment with extract from O. vulgare at a concentration of 250 µg/mL. The same concentration of the plant extract (250 µg/mL) killed 67% of the cells (33% of viable cells) in the treatment of MDA-MB-231 cell lines after 72 h. These results suggest greater effectiveness of the extract from O. vulgare on HCT-116 cell line.The results indicated more dramatic effects of cytotoxic activity after the treatment with extract in vitro cytotoxic activity. The effects of extract were expressed by IC50 values (concentration which inhibit 50% of cell growth), as a parameter for cytotoxity.O. vulgare is considered to be a particularly valuable source of effective anti-proliferative and cytotoxic substances. Previous research of O. vulgare in Serbia -2,5-diphenyltetrazolium bromide (MTT) were obtained from SERVA, Germany. All other solvents and chemicals were of analytical grade.Origanum vulgare L. were collected in Avgust 2010 from the region of Pčinja river gorge in south Serbia. The voucher specimen of O. vulgare was confirmed and deposited in Herbarium at the Department of Biology and Ecology, Faculty of Science, University of Kragujevac. The collected plant parts were air-dried in darkness at room temperature (20 °C). Aboveground plant parts of O. vulgare (10 g) was broken in the small pieces 2–6 mm by using a cylindrical crusher and extracted with methanol using Soxhlet apparatus. The extract was filtered through a filter paper  and evaporated. The residue was stored in a dark glass bottle for further processing.The prepared plant material from 2 at 37 °C. Cells were grown in 75 cm2 culture bottles supplied with 15 mL DMEM, and after a few passages cells were seeded in 96-well plate. All studies were done with cells at 70 to 80% confluence. HCT-116 and MDA-MB-231 cell lines were obtained from American Type Culture Colection. Cells were maintained in DMEM supplemented with 10% Fetal Bovine Serum, with 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were cultured in a humidified atmosphere with 5% COO. vulgare at different concentrations  for 24 and 72 h. Untreated cells served as the control. HCT-116 and MDA-MB-231 cells were seeded in a 96-well plate (10 000 cells per well). After 24 h of cells incubation, the medium was replaced with 100 μL medium containing various doses of methanolic extracts of 2 for 2–4 h. The colored crystals of produced formazan were dissolved in 150 μL DMSO. The absorbance was measured at 570 nm on Microplate Reader. Cell proliferation was calculated as the ratio of absorbance of treated group divided by the absorbance of control group, multiplied by 100 to give a percentage proliferation. After 24 and 72 h of treatment the cell viability was determined by MTT assay . The prot-test or the one-way ANOVA test for multiple comparisons. A p value < 0.05 was considered as significant. The magnitude of correlation between variables was done using a SPSS  statistical software package . The IC50 values were calculated from the dose curves by a computer program . The data is expressed as means ± standard errors (SE). Biological activity was examined in three individual experiments, performed in triplicate for each dose. Statistical significance was determined using the Student’s Origanum vulgare showed significant antiproliferative activity in both HCT-116 and MDA-MB-231 cell lines. Inhibitory activity appeared to be particularly conspicuous in treated HCT-116 cell lines, whereas at the highest concentration (500 µg/mL), only 10% of the cells remain viable. These data indicate high antiproliferative potential of biologically active substances extracted from Origanum vulgare.Based on these results, we conclude that the methanolic extract of


The in vitro growth inhibition of puerarin or 5-FU alone or combined on BGC-823 cells was determined using a cell counting kit 8 (CCK-8) on living cells. Apoptotic morphological features and proteins expression levels were detected by Hoechst 33258 staining, an Annexin V/propidium iodide apoptosis kit and western blot analysis, respectively. Tumor xenografts were established in nude mice and the inhibitory effects and side effects were detected. Results of the CCK-8, Hoechst 33258 staining and flow cytometry revealed that the combined treatment was more effective than the separate treatments. The tumor volume was 90.65% of that of the controls and the mean tumor weight was only 0.125 g at the end of the experiment in the combination group compared with the control group (0.822 g). In addition, it was determined that liver and renal toxicity did not increase in combined treatment. These findings showed that puerarin and 5-FU produced a significant synergic effect on gastric cancer cells, while there was no increase in side effects.Combination chemotherapy is a crucial method in the treatment of gastric cancer. The aim of the present study was to investigate the inhibitory effects of puerarin and 5-fluorouracil (5-FU) on BGC-823 gastric cancer cells  Gastric cancer (GC) is the fourth most frequent type of cancer around the world . In East21H20O9, was isolated from the herbal medicine Radix puerariae (The compound 5-fluorouracil (5-FU) remains a first line antineoplastic treatment in clinical practice . Howeveruerariae . Previouuerariae –9. Reseauerariae ,11. Howein vitro and in vivo.The aim of the present study was to evaluate the combined effects of puerarin and 5-FU on BGC-823 gastric carcinoma cells 2. Puerarin and 5-FU were obtained from Sigma-Aldrich  and were 98% pure by reverse-phase high-performance liquid chromatography.The BGC-823 human gastric cancer cell line was purchased from the Shanghai Cell Collection . The cells were maintained in DMEM  supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic solution  at 37°C in a humidified atmosphere of 95% air/5% CO5 per well. Following exposure to puerarin , 5-FU  or puerarin+5-FU for 48 h, cells were incubated at 37°C for another 4 h with CCK-8 (10 μl per well). Absorbance was measured at a wavelength of 490 nm using an iMark microplate reader . Each sample was subjected to three independent experiments.Cell proliferation was quantified with a Cell Counting kit-8 assay . Cells were suspended in a complete DMEM and subsequently seeded in 96-well microtiter plates at a density of 1×10m [fa/(1-fa)]1/m, where D is the dose, Dm is the median effect dose required to produce , fa is the fraction of the system affected by D, and m is a Hill-type coefficient signifying the sigmoidicity of the dose-effect curve. The combination index (CI) was obtained by using the Biosoft CalcuSyn software  written in BASIC for automatic graphing of the CI with respect to fa. When smaller than 1, equal to 1, or greater than 1, CI indicates synergism, summation, antagonism, respectively.As previously described ,13, the Detection of apoptotic morphological features was performed using Hoechst 33258 staining (Beyotime). Cells were cultured in a six-well plate and incubated with puerarin , 5-FU (80 μM) or puerarin+5-FU for 48 h. Then, 20 μM Hoechst reagent was added to each well with for 10 min at room temperature. Changes in nuclear morphology were observed with a fluorescence microscope . In each group, ten microscopic fields were selected randomly and counted.6 cells/μl. Then, 5 μl Annexin V-fluorescein isothiocyanate and 10 μl PI were added and the cells were incubated at room temperature for 5 min in the dark. At the end of the incubation period, cell apoptosis was analyzed on a FACScan flow cytometer .An Annexin V/PI apoptosis kit was used to quantify the percentage of cells undergoing apoptosis . Cells were incubated for 48 h with either puerarin, 5-FU or puerarin and 5-Fu. Following incubation, the cells were washed twice with cold phosphate-buffered saline and resuspended in binding buffer (500 μl) at a concentration of 1×10A wet transfer system was used to detect the protein levels. Proteins were resolved by SDS-PAGE  and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 10% non-fat dry milk and incubated with primary antibodies  overnight at 4°C. The following day, horseradish peroxidase-conjugated polyclonal goat anti-rat secondary antibodies  were added, and the cells were incubated for 2 h at room temperature. Then, the membranes were placed in an enhanced chemiluminescence system  and imaged in a dark room. The antibodies were used as follows: B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax) and GAPDH .6 cells/mouse) were injected subcutaneously into the right dorsal area. When tumors grew to a mean volume of 160 mm3, the mice were treated were as follows: i) Control, 0.9% saline; ii) puerarin group, 30 mg/kg/day puerarin, iii) 5-FU group, 12 mg/kg/day 5-FU; iv) puerarin+5-FU group, combination of puerarin (30 mg/kg/day) and 5-FU (12 mg/kg/day), three times a week for three weeks. Tumor volume (TV) and weight were recorded every three days. TV was calculated by the following formula: TV (mm3) = d2 × D/2, where d and D are the shortest and longest diameters, respectively. Following three weeks of treatment, the mice were sacrificed by orbital sinus bleeding and the tumors were segregated and weighed. Liver and renal injury were measured through detection of the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (Cr). The tumor tissues were harvested for pathological examination to confirm that they were the same type of tumor. The levels of apoptosis in the cells of the tumor xenograft were measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay according to the manufacturer’s instructions .Male BALB/c-nu/nu nude mice,  were caged in groups of six and fed with a standard diet and water ad libitum. The study was approved by the ethics committee of Remnin Hospital of Wuhan University. Cells . All data are expressed as the mean ± the standard deviation. The mean values of the different groups were compared using non-parametric analysis (Mann-Whitney rank sum test), and a value of P<0.05 was considered to be statistically significant.in vitro. As shown in After 48 h of drug exposure, growth of BGC-823 cells was clearly inhibited in a dose-dependent manner Following exposure to the drugs for 48 h, the inhibition rate was tested. The combination effects were studied by the method of Chou and Talalay . TreatmeApoptosis was one of the predominant types of programmed cell death which involved a series of biochemical events leading to specific cell morphology characteristics, including cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. As shown in In order to verify the apoptotic mechanism, the levels of the mitochondrial pathway proteins Bax and Bcl-2 were detected. As shown in The effect of puerarin and 5-FU on the growth of primary tumor xenografts in nude mice was examined. Tumor volume was recorded every three days. The volumes of the tumors in the treatment groups were clearly reduced compared with those in the control group, while the inhibition rate in the combination group was 90.65%, which was a more significant inhibition than that in the other three groups P<0.05; . The datAt the end of the experiment, 24 mice were necropsied to assess the side effects. There were no injuries to the liver or kidney observable with the naked eye. ALT, AST, BUN and Cr levels in the serum were detected to evaluate liver or renal injuries. The results were not indicative of any injury and there were no significant variations among the four tested groups P>0.05, .GC remains the most commonly diagnosed type of cancer worldwide, exhibiting a high annual mortality ,3. Earlyin vitro and in vivo.5-FU, which is widely used for the treatment of several human malignancies, has been used in clinical treatments for years . Howeveret al . These results showed that puerarin combined with 5-FU at appropriate concentrations had a synergistic effect on BGC-823 cells, compared with that of puerarin or 5-FU alone. Wang et al  examinedng et al  showed tng et al . Hien etl (et al  indicateIn conclusion, the present study indicated that low-dose 5-FU combined with puerarin inhibited cell viability more effectively, compared with 5-FU and puerarin alone. Puerarin may potentiate the antiproliferative effect of 5-FU and reduce the therapeutically required dose of 5-FU, without increasing the toxicity, which provided a valuable novel treatment for patients with GC.


Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc2155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc2155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc2155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells. Mycobacterium tuberculosis, is a slow-growing intracellular pathogen. To control mycobacterial infection, it is necessary to clarify the mechanisms of host–pathogen interactions and how the pathogen survives in host cells.Tuberculosis is an infectious disease worldwide, and the causative agent, Mycobacterium smegmatis is a rapidly growing nonpathogenic mycobacterium. Its features make it useful as a tool for genetic analysis in the research on mycobacterial infection. In particular, the M. smegmatis mc2155 strain is widely used because of its high rate of transformation. We screened another M. smegmatis strain, J15cs, and constructed a convenient and safe host-vector system of a mycobacterial model. We previously reported that the J15cs strain replicated our original vector, pYT923, and survived in host cells longer than the mc2155 strain [2155 strains. The J15cs strain lacks glycopeptidolipids (GPLs) on the cell surface, and this may affect its morphology and survival in host cells [Mycobacterium avium, M. intracellulare, M. scrofulaceum, M. abscessus, M. chelonae, and M. smegmatis. Structurally, GPLs comprise a lipotetrapeptide core, fatty acid, and oligosaccharide. Three amino acids  link a 3-hydroxy or 3-methoxy long-chain fatty acid (C26-34) to the N-terminal and an amino alcohol, alaninol, to the C-terminal. The 6-deoxy-talose is glycosidically linked to the D-allo-threonine and a methylated L-rhamnose to the alaninol to generate the non-specific core GPLs found in all members of GPL-producing mycobacteria. In M. avium and M. intracellulare, an oligosaccharide unit is elongated from 6-deoxy-talose, leading to the serotype-specific, so-called polar GPLs [GPL, also called C-mycoside, is produced in the outer layer of some non-tuberculous mycobacteria, including cid C26-3 to the Nlar GPLs .mps gene that involves the tetrapeptide synthesis in the GPLs [In this study, we examined the GPL biosynthesis pathway and clarified the causative gene cluster that deletes the GPLs in the J15cs strain. We targeted the the GPLs . InteresM. avium Jucho strain and maintained in our laboratory [2155 strain (ATCC 607) was purchased from the American Type Culture Collection . Both strains were grown on Middlebrook 7H11 agar  with 0.5% glycerol and 10% Middlebrook OADC enrichment (Difco Laboratories) or nutrient agar at 37°C for 2–7 days. Escherichia coli was cultured in LB broth. Kanamycin (20 μg/ml) was added to the selective medium for pNN2-containing plasmids.The J15cs strain is diverged from the boratory . The mc2mps1 and mps2 genes were amplified by polymerase chain reaction (PCR) with specific primer pairs. The primers were prepared in reference to the reported sequence of the mc2155 strain (DNA fragments containing the region covering the 5 strain . KOD FX mps1 gene of the mc2155 strain were amplified by PCR with specific primer pairs (Primer Nde-mps1 and Pac-mps1) in NdeI-PacI and ligated into the same sites of pNPP, resulting pNPP-MPS1. The pNPP is a pUC19 derivative containing the promoter region of aphII and the terminator of the antigen gene from Mycobacterium kansasii [XbaI and the inserted fragment was ligated into the same site of an E. coli-mycobacteria shuttle plasmid pNN2 to yield pNN-MPS1 [DNA fragments containing the whole kansasii . Then, ppNN-MPS1 . The conThe GPLs were detected by thin-layer chromatography (TLC) as described previously . BrieflyColony morphology was observed using an optical microscope after incubation for 10 days on nutrient agar plates. To prepare the bacteria for scanning electron microscopy (SEM), they were suspended with 0.1 M phosphate buffer (pH 7.2), passed through a 5 μm membrane filter  to remove aggregates, and then put on coverslips  coated with 0.1% (w/v) poly-L-lysine solution. The bacteria were fixed with 2.5% glutaraldehyde  in 0.1 M phosphate buffer for 2 h at room temperature, washed twice with 0.1 M phosphate buffer, and post-fixed with 1% osmium tetroxide (TAAB Laboratories Equipment) in 0.1 M phosphate buffer for 1 h at room temperature. The bacteria were then dehydrated in a graded ethanol series. After permutation with 3-methyl butyl acetate, the critical point drying was determined using a Hitachi HCP-2 Critical Point Dryer . SEM was performed at ×5,000 on a Hitachi FE-SEM S-4700 instrument (Hitachi High-Technologies Corp.) using an accelerating voltage of 10 kV .2. The J774.1 cells were grown to semi-confluence for 48 h on 24-well flat-bottom tissue culture plates . M. smegmatis strains were pre-cultured on nutrient agar plates for 7 days. PBS added on agar plate. Bacterial cells were suspended, and were mixed slowly. After collecting and pumping by 23G syringe, the bacterial suspension was passed through a 5 μm membrane filter to remove aggregates. A single-cell suspension was checked by acid-fast staining, and was prepared at a concentration of approximately 1 × 108 CFU/ml. A bacterial single-cell suspension of 1×107 CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed twice with PBS in order to remove non-infecting bacteria. To kill any remaining extracellular bacteria, the infected J774.1 cells were further incubated for 2 h with fresh culture medium supplemented with 5% inactivated FCS and 200 μg/ml of gentamicin . The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells were cultured for 8 days. The survival of mycobacteria was evaluated by counting colony-forming units (CFU). The adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The lysates plated on nutrient agar. The experiments were performed in triplicate, and statistical analysis was carried out using Dunnett’s test [A murine macrophage, J774.1 cell line , was cultured in RPMI 1640 medium  supplemented with 10% inactivated fetal calf serum  at 37°C in 5% CO2155 strain (GenBank accession no. AY439015) [2155 strain, whereas the mps2 sequence of the J15cs strain was identical to that of the mc2155 strain.The DNA fragments of MSMEG_0400–0402 were amplified by PCR and the sequence was determined. The nucleotide sequence MSMEG_0400–0402 of the J15cs strain was deposited in the NCBI GenBank database under accession number AB924538. The results showed a segment of DNA with a total length of 18,238 bps. The sequence was identical to that reported for the mcY439015)  except fmps1 of the mc2155 strain (mc2155-mps1) was inserted into a pNN2 vector  of the J15cs strain, which were complemented with the mc2155-mps1, were obtained. The phenotypes of GPLs were examined. The alkaline-stable lipids were analyzed by TLC and are shown in mps1-complemented J15cs mutants were restored with the same Rf (retention factor) values on TLC as those of the mc2155 strain. In addition to the comparative Rf value, the molecular weight of each spot was measured by MALDI-TOF/MS , and are responsible for synthesis of the peptidyl core of the GPLs [mps1 in the J15cs strain, and insertion of mc2155-mps1 into the J15cs mutant restored expression of the GPLs. The MALDI-TOF/MS analysis showed that the detailed structure, modifying groups of acyl chains, the sugar of GPLs, and the GPLs of the mps1-complemened J15cs mutant were completely identical to those of the mc2155 strain. These results implied that the mps1 mutation directly caused the deletion of GPLs in the J15cs strain. Because the deletion of 18 bps did not cause a frame-shift mutation, these deleted 6 amino acids in the Mps1 protein are considered to be important for the secondary, tertiary protein structures and active domain. Based on the phenotype of the mps1-complemented J15cs mutant, the deletion of GPLs affects two prominent features in the J15cs strain. One is bacterial size. The J15cs strain is longer than mc2155, and that of the mps1-complemented mutant was almost the same length as mc2155 rather than the parent J15cs strain. The other is the colony morphology. The J15cs strain was the rough type, and the complemented mutant shifted to the smooth type. These phenomena were due to the presence of GPLs.involved ,12. In tthe GPLs ,14. Compmps1-complemented J15cs mutant did not survive as long as the parent J15cs strain in host cells. This result showed that the survival of the J15cs strain is correlated with the lack of GPLs. Toll-like receptor (TLR) 2 recognizes GPLs [Mycobacterium abscessus is a rapidly growing mycobacteria closely related to M. smegmatis. This species is recognized as a pulmonary pathogen in humans. Recently, Bernut et al. reported that the rough variant of M. abscessus, which is devoid of GPLs, causes more severe clinical disease than the smooth variant. The mechanism of the increased virulence of the M. abscessus rough variant was explained to be the massive production of serpentine cords, which are absent in smooth variant infection. In apoptotic macrophages, the rough variant of M. abscessus initiated cord formation and grew so large that the macrophages and neutrophils could not phagocytize them [M. abscessus rough variant is immunostimulatory and invasive in macrophage infection [M. abscessus rough variant is associated with synthesis/exposure of the lipoprotein at the cell surface [2155 mutant strain exhibited rough colony morphology, and indicated that GPLs are involved in the cell wall permeability barrier of M. smegmatis [Next, we were interested in whether these phenotypes correlated with bacterial survival in host cells or not. Surprisingly, the zes GPLs ,17. The ize them ,19. In a surface . Etiennemegmatis . These pM. avium and M. intracellulare produce a serotype-specific polar GPL that plays a crucial role in the host immune responses. According to our previous report, the acetyl or methyl modifications of oligosaccharides are necessary for host cells to recognize polar GPLs via TLR2. The activities of the polar GPLs are greatly decreased by the deletion of acetyl groups from oligosaccharides [M. abscessus and the polar types in the M. avium-intracellulare complex. On the other hand, GPLs are proposed to confer the capacity for sliding motility, biofilm formation, and dormancy (to be published elsewhere) [As for the GPLs, the major nontuberculous pathogens charides . The majcharides ,24. In tsewhere) ,26.mps1 mutation causes the deletion of GPLs in the J15cs strain, and affects bacterial size, morphology, and survival in host cells.In this study, direct evidence was shown that the


The aim was to analyse and adapt the physical properties of the scanners and study variations of biological tumour volume (BTV) in early and late FET-PET.Imaging results from subsequent measurements  are compared. O- of the BrainPET was 2.63mm–3.47mm and 4.39mm–5.10mm for the HR+ (10mm off-centre) [41% of patients showed a considerable deviation between early and late FET-PET. BTV increased in 14 patients. Four patients showed a FET positive region only in late FET-PET.Taking into account the physical differences of PET scanners is important for cross-scanner studies. It was shown in a patient study that BTV may vary between early and late FET-PET, which is important for patient management and needs further investigation.


Alhagi sparsifolia is a typical desert phreatophyte and has evolved to withstand extreme dry, cold and hot weather. While A. sparsifolia represents an ideal model to study the molecular mechanism of plant adaption to abiotic stress, no research has been done in this aspect to date. Here we took advantage of Illumina platform to survey transcriptome in primary roots of A. sparsifolia under water stress conditions in aim to facilitate the exploration of its genetic basis for drought tolerance.A. sparsifolia seedlings in the course of 24h of water stress following 6h of rehydration. The resulting 38,763,230, 67,511,150, 49,259,804 and 54,744,906 clean reads were pooled and assembled into 33,255 unigenes with an average length of 1,057 bp. All-unigenes were subjected to functional annotation by searching against the public databases. Based on the established transcriptome database, we further evaluated the gene expression profiles in the four different primary roots samples, and identified numbers of differently expressed genes (DEGs) reflecting the early response to water stress (6h vs. 0h), the late response to water stress (24h vs. 0h) and the response to post water stress rehydration (30h vs. 24h). Moreover, the DEGs specifically regulated at 6, 24 and 30h were captured in order to depict the dynamic changes of gene expression during water stress and subsequent rehydration. Functional categorization of the DEGs indicated the activation of oxidoreductase system, and particularly emphasized the significance of the ‘Glutathione metabolism pathway’ in response to water stress.We sequenced four primary roots samples individually collected at 0, 6, 24 and 30h from the A. sparsifolia, thus providing a substantial contribution to the sequence resources for this species. The identified DEGs offer a deep insight into the molecular mechanism of A. sparsifolia in response to water stress, and merit further investigation.This is the first description of the genetic makeup of  Drought stress due to water scarcity has emerged as one of the most urgent issues facing terrestrial ecosystem in many areas of the world to date. Under water deficit conditions, plants often show an array of morphological, physiological and biochemical changes, which may negatively impact plant germination, growth, development as well as reproduction, leading to substantial losses in both natural ecosystem and agricultural production ,2. Thus,2O2), Ca2+, phosphatases . To this end, 929 up- and 362 down-regulated unigenes were detected from the P-6h library as compared to the P-0h library, and defined as the early water-stress inducible and repressed genes, respectively  as compared to 6h of rehydration. A total of 172 common genes were identified from the early and late water-stress inducible genes and the rehydration repressed genes, showing an ‘up-up-down’ expression pattern . ConversAs an objective means of generalizing the biological functions represented by the significant change in gene expression, the GO category enrichment analysis by using GOseq  were preLikewise, for 936 late water-stress inducible genes with GO annotations, a total of 32 significantly enriched GO terms were also predominantly clustered into biological process and molecular function. The ‘apoplast’ was the unique enriched subcategory under cellular component . Among tA. sparsifolia primary roots.In contrast to the relative narrow distribution of the early and late water-stress inducible genes in GO category enrichment, the early and late water-stress repressed genes were individually enriched into 43 and 41 GO terms clustered in all of the three main categories Figs.  and 10B,The rehydration inducible and repressed genes, however, showed a completely reverse pattern in GO category enrichment when compared to the water-stress inducible and repressed genes. As shown in A. sparsifolia primary roots during water stress and subsequent rehydration, the identified DGEs were further mapped to reference canonical pathways in KEGG database with KOBAS [S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGD), L-ascorbate peroxidase and spermidine synthase, which were represented by eight unigenes with the ‘up-up-down’ expression pattern, suggesting their dynamic expression pattern in the course of the water stress and the following rehydration. Besides the ‘Glutathione metabolism pathway’, four additional pathway, such as ‘Metabolism of xenobiotics by cytochrome P450’, ‘Drug metabolism-cytochrome P450’, ‘Alanine, aspartate and glutamate metabolism’ and ‘Microbial metabolism in diverse environments’, were also significantly enriched from the late water-stress inducible genes, suggesting the complexity of the biological pathways changes under long-term water stress.To ascertain the biological pathways that are active in th KOBAS , and comTo validate the differential expression data revealed by RNA-Seq, the RT-qPCR assay was performed on 10 unigenes which demonstrate the ‘up-up-down’ expression pattern in the course of 24h of water stress following 6h of rehydration  and one protein have been deposited in Genbank prior to Jan., 2015. Understanding of the molecular mechanism underlying the drought tolerance of A. sparsifolia is greatly impeded and delayed. In the current study, we first report the transcriptomic analyses conducted on primary roots of A. sparsifolia, and identify numbers of DEGs responding to the early water stress, the late water stress as well as the immediate rehydration after water stress, thereby offering an opportunity to view the sequence resources of A. sparsifolia, and in particular the dynamic profile of gene expression in its primary roots under water stress.nditions ,32. HoweA. sparsifolia. Interestingly, the ‘oxidation-reduction process’ remained dominant in the group of the rehydration repressed genes, suggesting that the active oxidoreductase system under water stress could be down regulated once under the well watered conditions. Nevertheless, the data together conclusively demonstrated the prime importance of the oxidoreductase system in primary roots of A. sparsifolia in response to water stress. In addition, consistent with the previous finding that sulfur is one of crucial factors determining abiotic stress tolerance in plant [A. sparsifolia primary roots. GO analysis also showed a strong enrichment of 12 unigenes associated with ‘cell wall biogenesis’ in the late water-stress inducible genes. This implied the occurrence of cell wall remodeling in A. sparsifolia primary roots upon water stress, providing further evidence that maintenance of cell wall integrity is essential for stress response [On the basis of GO category enrichment analysis of the DEGs, we observed that the ‘oxidation-reduction process’ present as a dominant term in both the early water-stress inducible genes and the late water-stress inducible genes. Given the essential role of the oxidoreductase system in stress response ,50, it win plant ,52, in tresponse ,54.as per NR and GO annotation, the genes within this dataset included proline dehydrogenase, serine/threonine protein kinase, MYB transcription factor, xyloglucan:xyloglucosyl transferase and pectinesterase. Regarding their unique expression pattern of ‘down-down-up’, these unigenes should represent the negative regulators of the water stress response in A. sparsifolia primary roots. In fact, proline dehydrogenase, as the first and rate-limiting enzyme of proline degradation, has been known to be repressed under water deficit but induced under hypoosmolarity [A particular interest was also given to the DEGs whose expression alternatively switched between induction and repression depending on water deficit or well watered conditions. We identified 172 unigenes, the expression of which was significantly up regulated at the early and late stages of water stress but repressed under the rehydration. This specific ‘up-up-down’ expression pattern strongly suggested their implication on positive regulation of the water stress response. Within this dataset, many unigenes were homologs of those have been well documented to function in biotic/abiotic stress tolerance in plants –59, suchmolarity ,61. Addimolarity –64.Gossypium herbaceum [A. sparsifolia primary roots seriously depended on the water supply conditions, indicating significance of the glutathione metabolism in A. sparsifolia primary roots against water stress.As far as is known that glutathione is a major substrate in antioxidative defense mechanisms by quenching reactive oxygen species, eliminating peroxides and scavenging free radicals –67. The erbaceum  and poplerbaceum . In this2O2 metabolism [A. sparsifolia primary roots depending on water conditions.It was worthy to emphasize that GST, G6PDH, 6PGD, L-ascorbate peroxidase and spermidine synthase, the five genes associated with the ‘Glutathione metabolism pathway’ showed the expression pattern of ‘up-up-down’. As reported previously, GST is one of the most important catalytic enzymes that utilize glutathione to function in the stress tolerance mechanisms of plant , and G6Ptabolism . Along wA. sparsifolia, a typical desert plant with extreme drought tolerance. A total of 33,255 unigenes were generated with a depth of 21.04 gigabase pairs, and 24,639 unigenes were functionally annotated with NR database. Based on this transcriptome database, we further explored the gene expression profiles in A. sparsifolia primary roots during 24h of water stress following 6h of rehydration, and defined numbers of unigenes specifically responding to water stress. The subsequent functional enrichment disclosed a large number of water-stress related genes associated with oxidoreductase system, and in particular highlighted the impact of glutathione metabolism on water stress, thus providing potential targets for candidate gene selection in improving the water stress tolerance of plants.This report represents the first application of Illumina sequencing technology for transcriptome studies in primary roots of S1 Fileq-value<0.005 and the absolute value of log2 (foldchange)>1 as the threshold. Table D in S1 File. Differentially expressed genes between P-24h and the untreated primary roots P-0h (late water-stress inducible and repressed genes). The significance of gene expression difference between samples was identified by using q-value<0.005 and the absolute value of log2 (foldchange)>1 as the threshold. Table E in S1 File. Differentially expressed genes between Rh-6h and P-24h (rehydration inducible and repressed genes). The significance of gene expression difference between samples was identified by using q-value<0.005 and the absolute value of log2 (foldchange)>1 as the threshold. Table F in S1 File. Common genes between the early and late water-stress inducible genes. Table G in S1 File. Common genes between the early and late water-stress repressed genes. Table H in S1 File. Genes exclusively expressed as the early inducible genes. Table I in S1 File. Genes exclusively expressed as the late water-stress inducible genes. Table J in S1 File. Genes exclusively expressed as the early water-stress repressed genes. Table K in S1 File. Genes exclusively expressed as the late water-stress repressed genes. Table L in S1 File. Genes showed ‘up-up-down’ expression pattern in the course of 24h of PEG-treatment following 6h of rehydration. Table M in S1 File. Genes showed ‘down-down-up’ expression pattern in the course of 24h of PEG-treatment following 6h of rehydration. Table N in S1 File. The significant KEGG pathways enriched from the early and late water-stress inducible genes (P-6h vs. P-0h) the early and late water-stress repressed genes (P-24h vs. P-0h), the rehydration inducible and repressed genes (Rh-6h vs. P-24h). The corrected p-value cut-off of 0.05 was set as the threshold, and only the enriched pathways with p-value less than 0.05 were shown.Table A in S1 File. Primers used in the RT-qPCR assay. Table B in S1 File. Statistics of unigenes in different expression-level interval. Table C in S1 File. Differentially expressed genes between P-6h and the untreated primary roots P-0h (early water-stress inducible and repressed genes). The significance of gene expression difference between samples was identified by using (ZIP)Click here for additional data file.


To fully exploit the possibilities of hybrid imaging a hybrid probe compatible with each imaging technology is required. Here, we present a hybrid nanoprobe for triple modality MR/SPECT/Fluorescence imaging. Our imaging agent is comprised of superparamagnetic iron oxide nanoparticles (SPIONs), labeled with 99mTc and an Alexa fluorophore (AF), together forming 99mTc-AF-SPIONs. The agent was stable in human serum, and, after subcutaneous injection in the hind paw of Wistar rats, showed to be highly specific by accumulating in the sentinel lymph node. All three modalities clearly visualized the imaging agent. Our results show that a single imaging agent can be used for hybrid imaging. The use of a single hybrid contrast agent permits simultaneous hybrid imaging and, more conventionally, allow for single modality imaging at different time points. For example, a hybrid contrast agent enables pre-operative planning, intra-operative guidance, and post-operative evaluation with the same contrast agent. Hybrid clinical imaging is an emerging technology, which improves disease diagnosis by combining already existing technologies. With the combination of high-resolution morphological imaging,  A single imaging modality cannot, by itself, give complete information of disease and the underlying molecular aberrations causing it. A combination of modalities may give the necessary information that physicians need to detect disease progression at an early stage, design patient-specific treatments, and monitor mization . For themization ,3,4. Multimodality imaging combines two or more imaging technologies in order to increase the diagnostic potential and to overcome the limitations offered by each single modality. A high-resolution imaging technology, providing morphological information, is typically combined with a high-sensitivity imaging technology sensing molecular aberrations. Examples of such systems include SPECT/CT, PET/CT, PET/MR, and MR/optical imaging modalities ,6,7,8. A®), angiography, and sentinel node mapping . The large surface to volume ratio also enables addition of different ligands for active targeting of, i.e., immune cells, angiogenesis or plaque [in vivo stem cell tracking and monitoring gene delivery [Superparamagnetic iron oxide nanoparticles (SPIONs) possess unique properties that make them well-suited for multimodality molecular imaging . Initialr plaque ,20,21,22delivery ,24. Labedelivery ,26,27,2899mTc-SPIONs) for early detection/staging of cancer by SPECT and MR [99mTc-SPIONs by labeling them with a fluorescent dye, and not only 99mTc and SPIONS for SPECT/MR, but also for optical fluorescence imaging. This triple-modality molecular imaging agent identifies the target by whole body SPECT and MR imaging, and highlight the target intra-operatively by fluorescence imaging. The radiolabeled and fluorescent SPIONs can also be visualized by microscopy during ex vivo pathological evaluation and the molecular targets can be quantified, e.g., by autoradiography. Here, we demonstrate that the attached dye does not affect the size and in vivo stability of the nanoparticles. The targeting ability of SLNs and biodistribution of the agent is demonstrated in Wistar rats. In our previous study, we suggested to use radiolabeled iron oxide nanoparticles  coated with polyethylene glycol and including primary amino groups. The coating prevents aggregation and enables functionalization. The SPIONs used in this study have been described elsewhere . BrieflyThe SPIONs  were labeled with the amino-reactive Alexa Fluor 647 (AF) carboxylic acid dye  by succinimidyl (NHS) ester coupling. The dye (10 mg) was dissolved in DMSO (1 mL) and incubated with the SPIONs for 2 h. The AF–SPIONs were washed three times with saline (0.9% v/v) and filtered after each wash by using a MACS MS column  and a strong cylindrical magnet. When the magnet was attached to the column, SPIONs got trapped in the filter while the fluid ran through. By removing the magnet, the nanoparticles could be eluted with saline. 99mTc. 99mTc-pertechnetate (99mTcO4−) was obtained from a 99Mo/99mTc-generator (DRN 4329 Ultratechnekow FM). The radiolabeling followed a previously developed protocol [99mTcO4− , which was kept in a vacuumcollecting vial. The hydrogen ion activity was adjusted to pH 4 by adding NaOH , after which, AF–SPIONs were added (0.1 mL) and gently shaken for 30 s. The mixture was incubated for 1 h at room temperature. The fluorescence and radiolabeled SPIONs (99mTc-AF–SPIONs) were filtered through MACS columns as described previously and eluted in saline . The remaining free amino groups on the SPIONs surface were used to label the nanoparticles with protocol  and was The radiolabeling yield was measured with instant-thin layer chromatography (ITLC). A small volume (0.8 µL) of reaction mixture was dropped on silica-gel impregnated glass fiber sheets  and aqueous methanol (85% v/v) was used as mobile phase for developing. The sheets were dried and cut in two strips, of 3 cm and 7 cm. The two segments were compared for radioactivity, as measured by a NaI(Tl) well type detector . 99mTc-AF-SPIONs, was filtered and pre-concentrated using MACS MS columns . The radioactivity of the buffer solution was compared to the radioactivity of the filtered 99mTc-AF-SPIONs. The reaction mixture, containing 99mTc-AF-SPION mixtures (10 µL) were diluted in human serum (500 µL) and incubated for 24 h at pH 7 in room temperature. The ITLC method was then used to measure any release of free 99mTc. SPIONs and 99mTc-AF-SPIONs were imaged with transmission electron microscopy  using 400 mesh carbon grids to measure the size of the nanoparticles and to evaluate possible aggregation. Two in vivo feasibility for imaging, two white Wistar rats weighing 250–300 g were used. The experiments were completed in compliance with local and national regulations approved by the Local Ethics Committee for Animal Research. The animals were slightly anesthetized with isoflurane and injected subcutaneously in the right hind paw, with 99mTc-AF-SPIONs (0.07–0.1 mL) dispersed in physiological saline buffer . The animals returned to their cages and, 5 h post injection, imaged with MR, SPECT and Optical modalities. To assess the activity distribution the animals were sacrificed and tissues/organs were measured in the NaI(Tl) well type detector .To show the 3; flip angle 90° and number of average 10. GRE: TE/TR 14.6/2000 ms; voxel size 0.8 × 0.8 × 1.3 mm3; flip angle 90° and number of averages 10.The animals were imaged with a 3T-MR system (Philips Achieva) equipped with a Sense-Flex-M coil. The images were acquired using Spin Echo (SE) and 3D Gradient Echo (GRE) pulse sequences with the following parameters. SE: TE/TR 80/3000 ms; FOV 100 mm; voxel size 0.5 × 0.5 × 1.3 mmA scintillation camera  captured anterior and posterior images in 15 min. The camera was equipped with a low energy, high-resolution parallel-hole collimator. SPECT/CT projections were acquired for 1 h, at 60 s per projection, with a 20% energy window centered at 140 keV.99mTc-AF-SPIONs was accomplished by a Helium-Neon laser . The laser source illuminated the animal with a slightly divergent beam with a spot size of approximately 2 cm2. The illumination spot was raster-scanned in an overlapping pattern across the region of interest, and one image was acquired for each illumination position. An air-cooled charge coupled device camera (Andor iXon DU-897) captured the image after having passed through a combination of a long-pass filter  and a dielectric band-pass filter centered at 670 nm. All images were background subtracted, subjected to a threshold, and summed to give the resulting image. The resulting image was overlaid a photograph of the animal to give an anatomical reference.The animals were shaved prior to imaging in an effort to minimize autofluorescence. Imaging of the animals was performed in epifluorescence mode. Laser induced fluorescence of the 99mTc-AF-SPIONs within the dissected organs was expressed in % of injected activity (% IA/g). The animals were dissected; the lymph nodes, kidneys, spleen, liver, and the hind paw were weighed and measured for activity in the NaI(Tl) well-type detector . The radioactivity measurement was corrected for background and decay. The uptake of the The lymph nodes were frozen, cut in 100 µm and 20 µm thick frozen coronal sections using a cryostat  and mounted on microscope slides and air dried at room temperature. The 100 µm sections were imaged and analyzed by digital autoradiography (DARG) with a silicon strip detector . Each second cryo-slice was hematoxilyn and eosin (H&E) stained and used as a reference for the DARG images. The 20 µm sections were DAPI stained and used for microscopy analysis (Zeiss Axiovert 200M). Advances in development of hybrid imaging technologies have created an enormous interest in the design and preparation of novel multimodality imaging agents ,30,31. Ain vivo toxicity. As the SPIONs are small in size and have a large area to volume ratio, a single nanoparticle can be labeled with a large number of radionuclides, fluorophores, and/or targeting ligands. In addition, polyethylene glycol coated SPIONs circulate in the blood pool/vasculature for a longer period of time compared with the low molecular weight gadolinium-based MR contrast imaging agents [99mTc-AF-SPIONs is crucial for targeting because it may influence the pharmacokinetic and biodistribution of the agent. In this work we propose combining information from MR, nuclear and optical images in order to have the full scale from whole-body imaging down to cellular and molecular imaging with use of one single agent. The major advantages to work with SPIONs are the easy production of the spherical core within narrow size range distribution, the simple coating procedure and the low g agents . The lab99mTc in order to evaluate the biodistribution of the agent in vivo. 99mTc is a generator produced isotope, highly available and the physical properties such as half-life and emitted energy is well-suited for imaging and quantification purposes. Despite the low cost of optical instrumentation and easy implementation for intra-operative surgery, there are only two fluorescent dyes (ICG and fluorescein sodium) approved by the FDA . In basi99mTc-AF-SPIONs, determined by ITLC, was 99%. 99mTc-AF-SPIONs remained at the origin whereas free 99mTc moved with the solvent front (Rf 1.0). Control measurements with 99mTc-pertechnetate have been performed to validate the method. Additionally, the high radiolabeling efficiency was confirmed by magnetic separation and found to be 99.7%. The radiolabeling yield of the 99mTc-AF-SPIONs incubated in serum showed a good dispersion and was found to be stable. After 24 h incubation of the 99mTc-AF-SPIONs, the amount of free 99mTc in human serum was less than 3%, analyzed with the ITLC method. The stability of the 99mTc-AF-SPIONs was also confirmed by the TEM images as demonstrated in 99mTc-AF-SPIONs is not affected by the labeling procedure; the spherical shape of the iron oxide core can be easily recognized and the surrounding polyethylene coating, which was shown to be intact. Both unlabeled  as well as in the renal (third) lymph node, 5 h post injection. The SPECT/CT images were transferred to a workstation where they were reconstructed and analyzed  and an in-house developed data visualization software  was used. The representative image clearly visualized the injection site and inguinal lymph node (right side) 99mTc–Alexa Fluor 647 labeled SPIONs. An accumulation of the fluorescence signal was observed and its location matches that of the inguinal lymph node. The injection site was not within the imaged region. Lymph nodes deeper than 2 cm have not been detected. However, intraoperatively, the agent showed to be useful to identify the SLN. 99mTc-AF-SPIONs stains the SLN green  was confirmed by the biodistribution analysis. The highest mean uptake was found in the inguinal LN (right side), 330 ± 120% IA/g, followed by the inguinal left side (150 ± 20% IA/g), renal LN side (100 ± 40% IA/g), spleen (1.3 ± 0.02% IA/g), kidneys (0.56 ± 0.08% IA/g) and liver (0.1 ± 0.2% IA/g) as presented in 99mTc-AF-SPIONs in SLN compared with our previous study using 99mTc-SPIONs (211 ± 225% IA/g) [The uptake of the 5% IA/g) . The hig99mTc-AF-SPIONs indeed localized to SLN. The fluorescence dye enabled localization and studies of microdistribution of the nanoparticles when the 99mTc radioactivity was no longer measurable. Furthermore, a non-homogenous uptake of the nanoparticles was found within the SLN as shown in (Digital autoradiography and microscopy showed that shown in . The nan99mTc-AF-SPIONs, for MR, SPECT and optical fluorescence imaging. The easy and straight forward labeling procedure and stability of the agent can be used for detection and intra-operative localization of the SLNs. In conclusion, we developed a new triple-modality imaging agent,


The QuantiFERON-TB Gold In-Tube (QFT-GIT) is a newly developed but widely used interferon-γ release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay’s ability to detect TB. We assessed the QFT-GIT diagnostic performance for active tuberculosis (ATB) in children and adults in an endemic country and explored the effects of glucocorticoids and anti-TB therapy on the diagnostic value of the QFT-GIT.A total of 60 children and 212 adults with suspected ATB were evaluated with the QFT-GIT. The association between the QFT-GIT diagnostic value and pretreatment factors was qualitatively and quantitatively assessed.The sensitivity of the QFT-GIT was 83.9% (95% CI 66.3%-94.6%) in children, and 73.7% (95% CI 57.8%-85.2%) in adults. Glucocorticoids affected the mitogen-stimulated response in both children and adults. In subjects undergoing glucocorticoid pretreatment, 25.0% of the children presented with false-negative QFT-GIT results, 28.6% of adults presented with indeterminate results. For subjects pre-treated with anti-TB drugs, 44.4% presented with false-negative QFT-GIT results.The QFT-GIT has higher sensitivity and specificity in children than adults. Glucocorticoid treatment negatively impacts the diagnostic value of the QFT-GIT in all age groups. Anti-TB treatment decreases the sensitivity of the QFT-GIT. Therefore, we recommend that the QFT-GIT assay be performed before TB-specific treatment is initiated and the test should not be used on people undergoing immunosuppression treatment, regardless of their age. A quantitative analysis of the QFT-GIT could be useful for assessing and monitoring TB-specific and non-specific immunity during conversion of the disease. Tuberculosis (TB) remains a serious public health threat. According to the latest World Health Organization report, there were 8.6 million new TB cases and 1.3 million deaths in 2012 [However, the high use of the Bacillus Calmette-Guerin (BCG) vaccination and the prevalence of latent TB infection (LTBI) can interfere with accurately diagnosing active TB (ATB). There are currently no optimal diagnostic criteria for ATB; each diagnostic method has limitations. The various clinical symptoms for ATB can lead to atypical chest X-ray presentations . BacteriMycobacterium tuberculosis (MTB)-specific antigen-stimulated IFN-γ production. MTB-specific antigens early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), both of which are encoded by the RD1 (region of difference 1) of MTB, are absent from BCG strains and most non-TB mycobacteria, so the specificity of IGRAs for detecting MTB infections is superior to that of the TST [T-cell-based IFN-γ release assays (IGRAs) have recently been developed. The two commercially available IGRA approaches are the whole blood-based QuantiFERON-TB Gold In-Tube (QFT-GIT) and the peripheral blood mononuclear cell (PBMC)-based T-SPOT.TB assay. ELISA (enzyme-linked immunosorbent assay) or ELISPOT (enzyme-linked immunospot assay) are used in IGRAs to detect  the TST , 10.While there have been numerous studies performed in adults, few studies have been conducted on the use of IGRAs in children. In an early study conducted in Germany, a country that stopped using BCG vaccination in 1998, the authors found that QFT-GIT sensitivity for diagnosing childhood TB disease is as high as 93%  . To inveA total of 272 subjects with clinically suspected ATB were enrolled in this study, including 60 children younger than 16 years of age who were admitted to the Shanghai Public Health Clinical Center and Children’s Hospital of Fudan University from December 2010 to August 2011, and 212 adults who were admitted to the Huashan Hospital of Fudan University between December 2010 and December 2011. The inclusion criteria are summarized in All subjects with suspected ATB at enrollment were classified into the following four groups according to their diagnosis after enrollment: 1) confirmed TB; 2) highly probable TB; 3) possible TB; 4) not TB . The catThe QFT-GIT assay  was performed according to the manufacturer’s instructions. A nil control tube, TB antigen  tube and mitogen tube (positive control) were used to collect whole blood from each subject (1ml/tube). All tubes were transferred to a 37°C incubator within 16 hours of collection. After a 16- to 24- hour incubation, the tubes were centrifuged, the plasma was removed and an ELISA was used to measure the IFN-γ (IU/mL) level. The level of IFN-γ release in response to MTB-specific antigens was calculated by subtracting the nil control tube from the TB antigen tube. The result was considered positive if the calculated result was at least 0.35 IU/mL and at least 25% more than the nil control value for any mitogen tube value. The result was considered negative if the positive control IFN-γ level minus nil control level was at least 0.5 IU/ml, and the calculated result was at least 0.35 IU/mL and less than 25% more than the nil control value, or no more than an additional 0.35 IU/ml. Indeterminate results were defined as those with a calculated result of less than 0.35 IU/ml, those with at least 0.35 IU/mL and less than 25% more than the nil control level and a positive control minus nil control value less than 0.5 IU/ml and those for which the nil tube value was more than 8.0 IU/ml. According to the instrument limitations, the upper limit of the test was 16 IU/ml and the lower limit was 0 IU/ml.2 test was used to compare proportions, and the Mann Whitney test was used to compare the IFN-γ level in different groups. P-values of less than 0.05 were considered to be statistically significant. All data were analyzed with SPSS 19.0 and GraphPad Prism 5.The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to evaluate the QFT-GIT assay. For sensitivity and specificity calculations, indeterminate results were included in the sum. The χThe ‘possible TB’ group, including 3 children and 15 adults, was excluded from the data analysis . The demThe qualitative results of the QFT-GIT assay for three diagnostic groups are listed in Of the patients with confirmed and highly probable TB, 38.7% (12/31) of the children and 36.8% (14/38) of the adults had been treated with glucocorticoids before the QFT-GIT test. Additionally, 16 adults (and no children) had received anti-TB therapy within 3 months before they were recruited; of these, 7 were also treated with glucocorticoids. To eliminate confounding factors in evaluating the effect of the two interventions on the assay performance, we removed the data from the 7 adults who underwent double interventions. Additionally, we stratified the remaining ATB patients into “no treatment”, “glucocorticoid therapy” and “anti-TB therapy” subgroups, and we recalculated the true positive and false negative rates for each subgroup. Compared to the “no treatment” subgroup, both therapy interventions had a negative effect on the test performance .There were a total of 19 indeterminate results. Of these, 73.7% (14/19) had therapy with glucocorticoids within the month preceding the QFT-GIT, 10.5% (2/19) were treated with glucocorticoids more than 1 month before the QFT-GIT, 5.3% (1/19) received both glucocorticoids and anti-TB treatment in the month preceding the QFT-GIT, 5.3% (1/19) were children, and 5.3% (1/19) lacked specific features . Low IFNWe found a significant difference (P<0.05) in the mitogen tube IFN-γ levels between patients treated with and without glucocorticoids in both adults and children . In the MTB and its components are potent inducers of monocyte cytokine production –14. T lyVarious factors affect the performance of the QFT-GIT ; Fig 2A.In the experiment, mitogen was used to activate all naïve and effector T cells in the tested blood. A significant decline in mitogen-stimulated IFN-γ release was found in both children and adults undergoing glucocorticoid therapy , suggestAfter removing subjects treated with either interventions (glucocorticoids or anti-TB therapy) from the analysis, mitogen-stimulated IFN-γ release was lower in the ATB patients for both age groups . The dir5 of the PBMCs. Even in the presence of down-regulation in either the average activation level or the number of activated T lymphocytes, as long as the number of mitogen panel spots is more than 20, T-SPOT.TB does not yield indeterminate results. With the low mitogen-stimulated IFN-γ production in the QFT-GIT, our data suggests that even in the case of immunosuppression due to glucocorticoids or other unknown reasons, there are rarely fewer than 20 mitogen-stimulated T cells. The insensitivity of the T-SPOT.TB for detecting fluctuations in the proliferation and activation of PBMCs reduces indeterminate results, favoring interpretations for immunocompromised patients.Young age has been correlated with indeterminate QFT-GIT results in several studies –22. HoweThis study consecutively enrolled hospitalized patients; therefore, bias could result from the unbalanced number of patients and the non-matched clinical characteristics that were compared between groups, including age, immune status, and therapeutic regimen. The small sample size for each group may have biased the analysis factors influencing the QFT-GIT. The preliminary findings in this study will be further validated in future randomized controlled trials.Both the MTB load and the T lymphocyte function influenced the QFT-GIT diagnostic value. IGRAs should be performed as early as ATB is suspected because the diagnostic value of these tests deceases as soon as anti-TB drugs are administered. In immunocompromised individuals with a higher likelihood of indeterminate results, the T-SPOT.TB is an alternative to the QFT-GIT. According to our quantitative analysis, the QFT-GIT can detect non-specific and TB-specific CMI in the development and progression of TB.